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nTU AM.05S FUN~iNa 14UMBIRSENVIRONMENTAL BIOTECHNOLOGY: MOVING FROM ThIE FLASK TO PE - 61102FTHE FIELD PR - 2312

- -. TA - A4B. AUTOB(S)GR - AFOSR-91-0046

JAM[ES W. BLACKBURN

1. PtRFORNInG Opt"MiZAT1ON NAME(S) AND AOORISS(ES) B.PERFORMING ORGANIZATION

University of Tennessee423 S. Stadium ,

Knoxville, TN 37996 AFOSRTR- 9 1

9. sPOMSOR~i MONTOR0NG AGENCY NAME(S) AND AOORESS4ES)1.SPSmOgjNLt Col Cerveny AGINCY REPOE NMUAFOSR/NLBuilding 410Bolling AFB DC 20332-6448

11. SUMUMENTARY NOTES

125. 0ISTRUU1ON /AVAMLAhUTrV STATEMENTIM iTOWNCa

Approved for public release; distribution unlimited

11. ABSTRACT (Mammum2O0*vWrJ

Environmental biotechnology for hazarduus wastes is uperaLiondiiv defined as the useof living organisms or their processes for socio-economnic benefit in environmentalprotection and restoration. Often, biotechnology for control of wastes and toxicmaterials is viewed as the extremes of either conventional biological waste treatmenttechnology or genetically engineered "super bugs" of consequent risk to the environ-ment.. Between these extremes, environmental biotechnology has evolved from theintegration of Engineering, Environmental and Biological sciences as an important newresearch field contributing to the development, application and optimization ofbiological processes in hazardous waste control. An analysis of applications ofbiological process in hazardous waste control leads to the identification of majorareas in which environmental biotechnology can contribute new problem solutions anddirectionc for the development or more reliable technology.

14SUATERMS 9 1-13074 I.MMAO AE

.17. Zi.Ja. AiWATON IL SECURITY -1t'FWCAtION it. SECUITY CLASSW"FITON IL MITN~ON A&STAOf REPOS? OF 11415 PAGE O. -"TkAeT

UNCLASSIFIED UNCLASSIFIED UNCLASSIFIEDUNITE

'45N7~dOO1.g0.SOO 9. 1(j1~ (w~St?.aae F@m 290 (Rev 2 69)

14

ENVIRONMENTAL BIOTECHNOLOGY: MOVING FROM THE FLASK TO THE FIELD

Special Final Report

Grant No. AFOSR-91-0046

tAugust 30, -199 j

Submitted by:

James W. Blackburn .

Principal Investigator

423 S. Stadium

University of Tennessee

Knoxville, TN 37996

Submitted to:

Lt Col T. Jan Cerveny

Program ManagerAFOSR/NL

Boiling AFB, Washington DC 20332-6449

Table of Contents

1 IN TR O D U C T IO N .................................................................................................. 1

2 P LA N N IN G ....................................................................................................... 2

3 P R O G RA M ....................................................................................................... 5

4 A B S T R A C T S ........................................................................................................ 104.1 PRESENTATION ABSTRACTS ................................................................. 10

4.1.1 SESSION I--ENVIRONMENTAL BIOTECHNOLOGY: CURRENTP E R C E PT IO N S ............................................................................................. 104.1.2 SESSION I--ENVIRONMENTAL BIOTECHNOLOGY: FIELD CASES T U D IE S ...................................................................................................... .. 184.1.3 SESSION Ill--TECHNICAL ISSUES AND CONCERNS IN ENVI-RONMENTAL BIOTECHNOLOGY IMPLEMENTATION ............................ 234.1.4 SESSION IV--NONTECHNICAL ISSUES AND CONCERNS INENV!RONMENTAL Z;OTECHNOLOGY IMPLEMENTATION ..................... 294.1.5 SESSION V--INTERNATIONAL ACTIVITIES IN ENVIRONMENTALBIOTECHNOLOGY .......................................... 37

4.2 POSTER ABSTRACTS ............................................................................... 41

5 F IN D IN G S .............................................................................................................. 5 5

1 INTRODUCTION

On October 17-19, 1990, a symposium entitled "Environmental Biotechnology--Moving From the Flask to the Field" was hosted at the University of Tennessee Confer-

ence Center in Knoxville, Tennessee. Over 300 individuals attended this meeting tohear and interact with forty Session Chairpersons, Invited Speakers anci Reviewers.

Sponsors included International Technology Corporation, American CyanamidCompany and the University of Tennessee Waste Management Research and Educa-tion Institute. The U.S. Air Force Office of Scientific Research was a sponsoring agcncyand the Oak Ridge Waste Management Association was a supporter.

The first symposium of its kind, the theme of the meeting was to merge an under-standing of the unique interests of waste generators, environmental service contractors,vendors, regulators, concerned citizens and th3 research community as they relate tothe performance of field bioremediation processes. The perceptions, state-of-the-artresearch and commercial applications, and both technical and non-technical problems

experienced in moving environmental biotechnology from the flask to the field were

addressed.

1i

2 PLANNING

The process of planning both the topics and structure of the symposium and theinvited speakers began with the actions of the Steering Committee. Members of the

Steering Committee included:

Bob Allen International Technology Corporation

James Blackburn Energy, Environment, and Resources Centcr,

The University of Tennessee, Knoxville

Bill Colglazier Waste Management Research and EducationInstitute, The University of Tennessee, Knoxville

Jimmy Cornette Environics Division, U.S. Air Force Engineeringand Services Center

John Corey Westinghouse/DOE Savannah River Site

Sue Markland Day Center for Environmental Biotechnology, The

University of Tennessee, Knoxville

Bob Fox International Technology Corporation

Tom Hayes Gas Research Institute

Ray Hillard Lederle Laboratories

Margaret Kelly Office of Solid Waste and Emergency

Response, U.S. Environmental Protection

Agency

Maureen Leavitt International Technology Corporation

Anthony Malinauskas Martin Marietta Energy Systems/DOE Oak

Ridge National Laboratory

Gary Sayler Center for Environmental Biotechnology, The

University of Tennessee, Knoxville

Godfred Tong Monsanto Company

J. Carroll Duggan Waste Technology Program,Valley Resource

Center, Tennessee Valley Authority

Dennis Wynne Technical Support Division, U.S. Army THAMA

2

The Steering Committee appointed James Blackburn the Symposium Coordinatorwith the responsibility of organizing the program and the sessions accordingly. Elaine

Keener of the University of Tennessee Conference Center was appointed ProgramManager and together with her staff, Elaine was responsible for the details of organizing

and holding the meeting. Gary Sayler was appointed the Proceedings Coordinator andhad responsibility for producing a proceedings volume entitled Environmental Biotech-

noloqy for Waste Treatment (G. S. Sayler, R. Fox and J. W. Blackburn, eds., PlenumPress, New York, 1991).

The program was divided into sessions and Session Chairs were appointed. The

Session Chairs had primary responsibility in contacting, inviting, and scheduling thespeakers nominated by the Steering Committee. The Session topics and Chairs are as

follow:

Session I-Environmental James Blackburn, The University of Tennessee

Biotechnology: Current

Perceptions

Session II-Environmental Jimmy Cornette, Environics Division, Air ForceBiotechnology, Field-scale Engineering and Service Center

Case Studies

Session Ill-Technical Issues Arthur Day, Bechtel Environmental, Inc. and

and Concerns in Environ- Gene Bowlen, Rutgers Universitymental Biotechnology

Implementation

Session IV-Nontechnical Sue Markland Day, The University of TennesseeIssues and Concerns in and Margaret Kelley, OSWER, U.S. EPA

Environmental Biotechnol-

ogy Implementation

Session V-International David C. White, The University of TennesseeActivities in Environmental

Biotechnology

3

Session VI-Symposium Robert Goldstein, Electric Power ResearchReview, Analysis, and InstituteDiscussion

4

3 PROGRAM

WEDNESDAY, OCTOBER 17,1990

WELCOME AND OPENING REMARKS Bill Colglazier, Director, Waste Management

Research and Education Institute, The University of Tennessee, Knoxville.

SESSION I - Environmental Biotechnology: Current Perceptions

INTRODUCTORY COMMENTS Chair: James Blackburn, Ph.D., Associate Director,

Energy, Environment and Resources Center, The University of Tennessee, Knoxville.

ENVIRONMENTAL BIOTECHNOLOGY: PERCEPTIONS, REALITY, AND APPLICATIONS

Gary S. Sayler, Ph.D., Director, Center for Environmental Biotechnology, The University

of Tennessee, Knoxville and Robert D. Fox, Distinguished Technical Associate, Interna-

tional Technology Corporation.

MEDIA IMAGES OF ENVIRONMENTAL BIOTECHNOLOGY: WHAT DOES THE PUBLIC•SEE9 Mike R. Fitzgerald, Ph.D., and Amy S. McCabe, Ph.D., Energy, Environment, and

Resources Center, University of Tennessee, Knoxville and Vanderbilt University Institute

tor Public Policy Studies.

PERSPECTIVES ON BIOREMEDIATION IN THE GAS INDUSTRY David G. Linz, Gas

Research Institute, Edward F. Neuhauser, Ph.D., Niagara Mohawk Power Company,

and Andrew C. Middleton, Ph.D., Remediation Technologies, Inc.

CONSIDERATIONS IN THE SELECTION OF ENVIRONMENTAL BIOTECHNOLOGYAS

VIABLE IN FIELD-SCALE WASTE TREATMENT APPLICATIONS Pat Taylor Woodyard,

CH2M-Hill, Inc.

THE TECHNICAL, ECONOMIC AND REGULATORY FUTURE FOR BIOREMEDIATION- AN

INDUSTRY PERSPECTIVE Keith Kaufman, President, Applied BioTreatment Association

and Vice President, Thorne Environmental, Inc.

REMOVING IMPEDIMENTS TO THE USE OF BIOREMEDIATION AND OTHER INNOVA-TIVE TECHNOLOGIES Walter Kovalick, Ph.D., Director, Technology Innovation Office,

U.S. Environmental Protection Agency.

BIOREMEDIATION RESEARCH ISSUES John H. Skinner, Ph.D., Deputy Assistant

Administrator, Office of Research and Development, U.S. Environmental Protection

Agency.

SESSION II - Environmental Biotechnology Field-Scale Case Studies

INTRODUCTORY COMMENTS Chair: Jimmy Cornette, Ph.D., Senior Scientist, Environ-

ics Divison, U.S. Air Force Engineering and Services Center.

BIOREMEDIA TION OF THE FRENCH LIMITED SUPERFUND SITE--FEASIBILITY STUDIES

TO THE CONSENT DECREE R. E. Woodward, Ph.D., Vice President, Bioremediation

and R. K. Ramsden, Ph.D., ENSR Corporation.

EVALUATION OF BIOREMEDIATION IN A COAL COKING WASTE LAGOON Maureen

Leavitt, Project Technical Coordinator, International Technology Corporation.

EVALUATION OF COMMERCIAL BIOREMEDIATION IN ALASKA Val Kelmeckis, Director

of Technology Evaluation, National Environmental Technology Applications Corporation.

FULL-SCALE REMEDIATION OF CONTAMINATED SOIL AND WATER Geoffrey

Compeau, Ph.D. and William D. Mahaffey, Ph.D., ECOVA Corporation. Lori Patras,

Unocal Corporation.

Panel Discussion

WHAT WILL THE BIOREMEDIATION BUSINESS BE LIKE IN 1995? Moderator: Edgar

Berkey, Ph.D., Executive Vice President, National Environmental Technology Applica-

tions Corporation.

THURSDAY, OCTOBER 18. 1990

SESSION I/l - Technical Issues and Concernsin Environmental Biotechnology Implementation

INTRODUCTORY COMMENTS Cc-Chairs, Arthur Day, Technical Integration Manager

for ORNL's RI/FS Project, Bechtel Environmental, Inc. and Gene Bowlen, Department of

Chemical and Biochemical Engineering, Rutgers University.

FEASIBILITY AND OTHER CONSIDERATIONS FOR USE OF BIOREMEDIATION IN SUB-

SURFACE AREAS Karolyn L. Hardaway, Ph.D., Texas Eastman Company

INTEGRATION OF BIOTECHNOLOGY TO WASTE MINIMIZATION PROGRAMS Godfred

E. Tong, Ph.D., Manager Process Technology, Corporate Research, Monsanto

Company.

BIO-REMEDIATION OF EXPLOSIVES CONTAMINATED SOILS (SCIENTIFIC QUESTION-

S/ENGINEERING REALITIES) Capt. Craig A. Myler, Ph.D., Project Officer and WayneSisk, U.S. Army Toxic and Hazardous Materials Agency.

PRACTICES, POTENTIAL, AND PITFALLS IN THE APPLICATION OF BIOTECHNOLOGY

TO ENVIRONMENTAL PROBLEMS Carol D. Litchfield, Ph.D., Senior Vice President,

Environment America, Inc.

WHAT IS THE Km OF DISAPPEARASE? Ronald Unterman, Ph.D., Vice President,

Research and Development, Envirogen, Inc.

USE OF TREATABILITY STUDIES IN DEVELOPING STRATEGIES FOR CON TAMINATED

SOILS Michael J. McFarland, Ph.D., and Ronald C. Sims, Ph.D., Division of Environmen-

tal Engineering, Utah State University, and James W. Blackburn, Ph.D., AssociateDirector, Energy, Environment and Resources Center, rhe University of Tennessee,

Knoxville.

BIODEGRADATION OF MIXED SOLVENTS BYA STRAIN OF PSEUDOMONAS Jim C.

Spain, Ph.D., U.S. Air Force Engineering and Service Center.

7

SESSION IV - Nontechnical Issues and ConcernsLnfEnvironmental Biotechnology Implementation

INTRODUCTOPY COMMENTS Co-Chairs: Sue Markland Day, Environmental Biotech-nology Policy Specialist, The University of Tennessee, Knoxville and Margaret Kelly,

Office of Solid Waste and Emergency Response, U.S. Environmental Protection Agency.

THE FIELD IMPLEMENTATION OF BIOREMEDIATION: AN EPA PERSPECTIVE Fran

Kremer, Ph.D., Senior Environmental Engineer, Office of Research and Development,

U.S. Environmental Protection Agency.

A HISTORICAL PERSPECTIVE: DOES GOOD PRESS AND/OR GOOD SCIENCE

GENERATE DEMAND? Thomas Zitrides, President, BioScience Management, Inc. andVice President, Applied BioTreatment Association.

WAYS TO IDENTIFY AND OBTAIN RIGHTS T-) TECHNOLCGY FROM FEDERAL FACILI-

TIES Jchn C. (Jack) Corey, Ph.D., Manager of Technology Transfer, Westinghouse

Savannah River Site.

AN OVERVIEW OF CURRENT ATTITUDES ON THE USE OF BIOTREATMENT FOR

CLEANUPS' William J. Lacy, Dr.Sc., P.E., D.E.E., President, Lacy & Company.

VIEWS OF A PROJECT MANAGER: WHAT ARE THE CRITICAL FACTS NECESSARY TO

WIN THE CONFIDENCE OF THE STATE REGULATOR? Frank R. Peduto, P.E., Senic

Engineer, New York State Department of Environmental Conservation.

IS THERE A NEED FOR A BIOREMEDIATION SPECIALIST CERTIFICATION? Morris A.

Levin, Ph.D., Program for Public Issues in Biotechnology, University of Maryland.

BIOLOGICAL MONITORING TO DEMONSTRATE CLEANUP ACTIONS Carl Gehrs,Ph.D., Environmental Sciences Division, Oak Ridge National Laboratory.

FEDERAL REGULATIONS: HOW THEY IMPACT RESEARCH AND COMMERCIALIZATION

OF BIOLOGICAL TREATMENT Sue Markland Day, Center for Environmental Biotechno-

ogy, University of Tennessee, Knoxville.

FRIDAY, OCTOBER 19, 1990

SESSION V - International Activities in Environmental Biotechnology

INTRODUCTORY COMMENTS Chair, David C. White, M.D., P').D., Distinguished Scien-

tist, The University of Tennessee, Knoxville.

POLLUTED HETEROGE' .E )/_S ENVIRONMENTS: MACRO-SCALE FLUXES, MICRO-

Sfl4LE MECHANISMS AND MOLECULAR-SCALE CONTROL Prof. G. Hamer, Ph.D.,Institute of Aquatic Science and Water Pollution Control, Swiss Institute of Technology,

Zurich, Switzerland.

THE PILOT PLANT TESTING OF THE CONTINUOUS EXTRACTION OF RADIONU-

CLEIDES USING IMMOBILIZED BIOMASS Marios Tsezos, Ph.D, Department of

Cnemical Engineering, McMaster University, Hamiliton, Ontario, Canada and RonaldMcCredy, Ph.D, Director of Biotechnology, CANMEI, Canada.

RESEARCH AND DEVELOPMENT PROGRAM FOR BIOLOGICAL HAZARDOUS WASTE

TREATMENT IN THE NETHERLANDS Esther R. Soczo, Coordinator for Development ofRemedial Action Technologies for Contaminated Soils and Deputy Chief of the Depart-

ment of Environmental Technology, National Institute of Public Health and Envircnmen-

tal Protection, RICM, Bilthoven, Netherlands.

SESSION VI - Symposium Review, Analysis. and Discussion

INTRODUCTORY COMMENTS Chair, Robert Goldstein, Program Director, Electric

Power Research Institute.

REVIEW, ANALYSIS, AND DISCUSSION Al Bourquin, Ecova Italia- Thomas W. Federle,

Proctor and Gamble, Ivoryate Tecnnical Center; C. P. Leslie Grady, Ciemsnon Univer-

sity; and William D. Mahaffey, Ecova Company.

9

4 ABSTRACTS

4.1 PRESENTATION ABSTRACTS

4.1.1 SESSION I--ENVIRONMENTAL BIOTECHNOLOGY: CURRENT PER-

CEPTIONS

Environmental Biotechnology: Perceptions, Reality and Applications, Gary S.

Sayler, Ph.D., The University of Tennessee, Knoxville and Robert D. Fox, Interna-

tional Technologies Corporation, Knoxville, TN

Environmental biotechnology for hazardous wastes is operationally defined as

the Use of hlnO, . Is. , orth,,r processes for sucio-economic benefit in envi-ronmental protection and restoration. Often, biotechnology for control of wastes

and toxic materials is viewed as the extremes of either conventional biological

waste treatment technology or genetically engineered "super bugs" of conse-

quent risk to the environment. Between these extremes, environmental biotech-nology has evolved from the integration of Engineering, Environmental and

Biological sciences as an important new research field contributing to the

development, application and optimization of biological processes in hazardous

waste control. An analysis of applications of biological process in hazardous

waste control leads to the identification of major areas in which environmental

biotechnology can contribute new problem solutions and directions for the devel-

opment or more reliable technology.

Media Images of Environmental Biotechnology: What Does the Public See?Michael R. Fitzgerald, Ph.D. and Amy S. McCabe, Ph.D., Energy, Environment,

and Resources Center, The University of Tennessee, Knoxville and Vanderbilt

University Institute for Public Policy Studies

Environmental biotechnology, as a method for effective and economical treat-

ment of hazardous wastes, is one of the most recent applications of biotechnol-

ogy to a major societal problem. Research and development in this area

continues apace, though fundamental policy questions relating to the successful

transfer of technology from laboratories to the private sector remain unanswered.

10

How, and to what Pxtnr, the government should regulate environmental biotech-

nology, and educate and inform the public are critical issues that public policy

makers must address.

The national television news plays a key role in biotechnology policy direction

and redirection in the United States. By communicating biotechnology informa-

tion directly to a mass audience, network news can educate viewers about topics

where a majority of Americans would not otherwise be attentive. Television news

is also influential because of its powerful potential to shape public opinion about

biotechnology applications. Finally, by featuring biotechnology stories, news

segments often emnhasi7 a critical lack of research support dedicated to con-

ceivably promising technologies. By controlling the flow of information--the

context in which it is presented, its position in the broadcast, and the amount of

time devoted--the news makes a wide-reaching statement about the relative

importance of biotechnology issues.

This presentation examines how environmental biotechnology has been por-

trayed by major network evening news organizations. Based on a systematic

examination of taped newscasts obtained from the Vanderbilt University

Television News Archive, the study illustrates how biotechnology has evolved, if

and how environmental biotechnology coverage differs from that of other bio-

technology applications, and what public perceptions are likely to be fostered by

the mass media portrayal of biotechnology issues. Implications for environmental

biotechnology policy are given.

Perspectives on Bioremediation in the Gas Industry David G. Linz, Gas

Research Institute, Edward F. Neuhauser, Ph.D., Niagara Mohawk Power Corpo-

ration, and Andrew C. Middleton, Ph.D., Remediation Technologies, Inc.

While there may be several applications for bioremediation in the gas industry,

tne nne of most immediate interest deals with the cleanup of manufactured gas

plant (MGP) sites. These plants operated in the U S. from 1816 to the 1960's,

producing manufactured or 'town" gas from coal and oil used for lighting,

cooking, and some industrial purposes. One of the by-products of gas produc-

11

tion was tar and other organic residues which can be currently found in the soil

and groundwaters at many MGP sites. Since 1986 the Gas Research Institute""nl) a;ong with individual gas companies have been sponsoring researchfocused on improving investigative and remediation methods for MGP sites. Earlyon it was demonstrated that contaminated groundwater from these sites could bebioremediated in municipal activated sludge systems along with municipal waste-

water. The next area of opportunity for bioremediation to be examined was thetreatment of soils contaminated with organic residues.

The ideal process for treatment of such soils would be one that consistentlyand predictably destroys a high percentage of the tar contaminants, i.e. polynu-clear aromatic hydrocarbons (PAHs), at a low cost without creating any otheradverse by-products. Testing of the treatment technologies has shown thatthermal processes, e.g. incineration, consistently and predictably destroy greaterthan 99% of the PAHs, but are costly. Conversely, testing has also shown thatbioremediations may degrade PAHs at a low cost but not consistently and pre-

dictably. A key need for the gas industry is a rapid, low-cost means to reliablyevaluate the effectiveness of bioremediation for MGP residues. Further, once itcan be shown that the residues are treatable using bioremediation, it must be

demonstrated that full scale systems can be designed and the performancereliably predicted and controlled.

In numerous bioremediation tests of soils from different MGP sites, resultshave shown the level of PAH treatment varying from zero to 90% with endpoints

for PAH ranging less than 10 to greater than 1000 ppm PAH. An analysis of these

tests has shown that soil type appears to be a major factor in these inconsistentresults. Soils which yield the higher endpoint concentrations of PAHs are theones containing higher amounts of clay and organic matter and/or significant

amounts of free tar. Bioremediation technology appears to be limited for thosewastes which have a high affinity for adsorption on soil particles and perhaps for

soils with high clay and/or organic matter.

A testing protocol has been developed to provide more rapid estimation ofbioremediation treatment endpoints. It is based in part on desorption isotherms

which determine the degree to which contaminants such as PAHs partition to the

12

soil water, and hence become bioavailable. The remaining PAHs are highly

bound to the soil matrix and are not bioavailable in bioremediation processes.Consequently, they are also not mobile in the environment and pose substantiallylower risks. Thus, although not able to produce consistent and predictabledestruction of all PAHs, bioremediation may consistently produce an environmen-tally acceptable treatment endpoint. Further data on the environmental behavior

of these treated residues is needed to convince the gas industry and regulatory

agencies of the validity of this approach.

Widespread acceptance among the regulatory community that a bioreme-diated soil is environmentally acceptable independent of the endpoint concentra-tion of PAHs has not been achieved. Often it is very difficult to obtain a fair

consideration of bioremediation with the certainty of the performance ofincineration looming in the background even in spite of the substantially higher

costs and the hurdles encountered in obtaining air emissions permits. This situa-tion epitomizes one of the greatest limitations to widespread use of bioremedi-ation. Its resolution is essential to the future implementation of bioremediation ofsoils at MGP sites and most likely at other industrial sites.

The next area of opportunity for bioremediation at MGP sites is the in situbioremediation of soils and groundwater. This process offers the potential forenvironmentally sound, cost-effective remediation of deeper contaminationmaking it very attractive to the gas industry. However, significant development

and demonstration will be required before it can be widely applied at MGP sitesas many technical and regulatory issues remain.

Considerations In The Selection of Environmental Biotechnology as ViableIn Field-Scale Waste Treatment Applications Patricia Taylor Woodyard, Indus-trial Hazardous Waste Services, CH2M Hill, Inc.

The selection of environmental biotechnology for field-scale implementation atcontaminated sites results from an identification, evaluation and weighing theimpact of numerous issues. This technology's technical viability is often weighed

against non-technical issues and the risks associated with its implementation.

13

The technology is often then compdred to issues anu risks arising from imple-menting alternative technologies at field-scale. The non-technical issues underconsideration often include the level of uncertainty associated with thetechnology in combination with legal, regulatory, sociopolitical, and businessimplications. Any issue or combination of issues can outweigh the potential tech-nical feasibility of environmental biotechnology. Identifying and evaluating theconsiderations and implications associated with t[.,,e technical andnon-technical issues under site remediation circumstances is a valuable step indetermining the viability of environmental biotechnology. "Uncertainty" becomes akey component that can influence both the technical and non-technical evalu-

ations.

The level of uncertainty with biotechnology's application under site specific

conditions can affect the selection of this technology. The uncertainty of the tech-nology's technical feasibility, irrespective of its application on a specific site, is

compounded by the uncertainty once the technology is applied to site specificsubsurface conditions. These uncertainties can in turn affect the viability of bio-technology's application on a site when combined with other technical and non-technical issues. This paper will describe these issues and associatedconsiderations that can affect the application of environmental biotechnology

under field-scale conditions.

The Technical, Economic, and Regulatory Future for Bioremediation: AnIndustry Perspective A. Keith Kaufman, M.S., BIOTA Division, Thorne Environ-mental, Inc., and Applied Bio-Treatment Association

The Biotreatment industry has undergone major evolutionary changes overthe last decade. From a little known, poorly understood approach to environmen-tal cleanup, bioremediation has rapidly developed into a widely accepted form of

remedial technology for a variety of environmental pollutants. Whereas thenumber of companies offering biotreatment in various forms five years ago couldbe counted on two hands, current estimates indicate that nearly 200 such com-panies now operate in the United States alone. Much of this rapid growth is due

14

to increased reports of successful bioremedial cleanups, as well as an increased

understanding among industry, regulators, and the public of the operational prin-

ciples associated with the technology.

While hope remains for continued growth and utilization of biotreatment, manyissues which could preclude the economic as well as practical benefits asso-ciated with the technology currently face the industry. So critical are these issuesthat unless strong countermeasures are adopted and implemented, the survival

of the biotreatment industry will be in serious jeopardy.

This presentation will focus on those issues which the industry believes posethe greatest threat to the continued development and utilization of bioremedia!

technology. Major emphasis will be placed on federal regulatory constraintswhich could undermine the very technology EPA has publicly supported. The sig-nificant economic and technical implications of these regulatory developmentswill also be explored. Efforts presently underway by the biotreatment industry to

mitigate these issues will also be addressed, as will developing criteria for the

registration of certified biotreatment specialists, a move designed to buildindustry credibility by documenting bioremedial competency of vendor compan-

ies.

Removing Impediments to the Use of Bioremediation and Other InnovativeTechnologies Walter W. Kovalick Jr., Ph.D., Technology Innovation Office, Office

of Solid Waste and Emergency Response, U.S. Environmental Protection Agency

The Office of Solid Waste and Emergency Response has set as one of itsgoals increased diversity of technologies used to remediate contaminated soilsand ground water. There are statutory and economic reasons to move awayfrom conventional methods such as stabilization, containment and incineration.Impediments exist however, to the application of any innovative technology.These include inhibiting regulations, conservative attitudes and fear of risk, and

lack of information on performance and cost. The Agency is addressing these

15

impediments through several avenues. Bioremediation is of special interestbecause of the potential for cost-effectiveness and permanence compared to his-

torical technologies.

We are pursuing changes in the implementation of the Resource Conservation

and Recovery Act (RCRA) to reduce obstacles to the use of innovative technolo-gies at Correcti,. a Action sites and Superfund sites. Policy changes are also

underway to make it acceptable to "fail" during the first applications of a new

technology. Finally, we are making efforts to increase our knowledge of perform-ance and cosc through a Bioremediation Field Initiative -- an evaluation of actual

treatment systems now operating at sites.

Bioremediation Research Issues John H. Skinner, Ph.D, Research and Devel-

opment, U.S. Environmental Protection Agency

Biological treatment is an effective remedial technology for managing liquidand solid hazardous wastes in above-ground reactors and in-situ systems. Over

the past several years EPA has had a number of successful bioremediation appli-cations to emergency responses such as oil contaminated beach clean ups from

spills, and Superfund remedial action clean-ups for sludges containingpolychlorinated biphenyls, volatile organics and metals, as well as soils contam-inated with creosote and poly aromatic hydrocarbons.

On February 22, 1990, EPA conducted a meeting on the Environmental Appli-cations of Biotechnology. Administrator Reilly and four EPA Assistant Administra-

tors, as well as close to 150 senior representatives from Federal/stategovernment, industry and academia attended the one day workshop. The

purpose of the meeting was to prepare a biotechnology agenda for action for the

1990's. Most of the discussion at this meeting had to do with bioremediation. Thefindings, recommendations, and proposed actions from the EPA-Industrymeeting were divided into six areas: field tests and demonstrations, technology

transfer, education and training, policy and regulations, research and pollution

prevention.

16

A joint Federal agency, academic and industry committee was established toimplement the actions from the February meeting. Some examples of theseactions include:

Field Test and Demonstrations -

- EPA is developing guidance for bioremediation treatability tests and demon-

strations.

• EPA will work with industry to conduct additional field tests of bioremediationat Superund, RCRA corrective action and underground storage tank sites.

* Working jointly with the State of Alaska and Exxon, EPA plans to coordinateadditional bioremediation field tests in Prince William Sound, Alaska on biologicaloil degradation on contaminated beaches.

Policy and Regulations -

• EPA's Office of Solid Waste and Emergency Response is considering a moreproactive policy statement regarding the use of innovative technologies (incl-

uding bioremediation) under Superfund as well as its other programs.

* EPA may be asking for additional bioremediation performance data for theproposed Land Disposal Restriction Regulations for soil and debris.

• States may be establishing bioremediation requirements for clean up of statelead hazardous waste sites.

17

Research -

• EPA is developing a research initiative for 1992 that will expand its existingresearch program to address critical gaps in the science of bioremediation.

- The critical role of academic Institutions in conducting research on bioremedi-ation will be reviewed and may be expanded.

4.1.2 SESSION II--ENVIRONMENTAL BIOTECHNOLOGY: FIELD CASE

STUDIES

Bioremediation of the French Limited Superfund Site - Feasibility Studies tothe Consent Decree R.E. Woodward, Ph.D. and D.K. Ramsden, ENSR Consult-ing and Engineering

In situ bioremediation of mixed, hazardous waste was demonstrated in a 0.5acre portion of the 7.3 acre lagoon. The petrochemical and other inaustrial wastesludges contained volatiles, polynuclear aromatic hydrocarbons, chlorinated

solvents, and PC priority pollutants in a viscous, hydrophobic, tarry matrixunder 3 to 15 feet of water. The growth and metabolic activity of microorganisms

native to the site was stimulated by a four phased mixing approach and by pro-viding a suitable pH, essential nutrients and oxygen. Priority pollutant concentra-tions, wastewater treatment parameters and toxicity were monitored to control

operations and to document the progress of bioremediation.

The demonstration confirmed the feasibility of in situ bioremediation and led toone of the first U.S. EPA Record of Decisions to use in situ bioremediation forcleanup of a large superfund site. A consent decree outlining the site remedial

action plan was signed by the PRP task group and published in the federalregister. This represents a landmark project for in situ bioremediation and hasestablished precedence for use of this technology at CERCLA and RCRA sites

nationwide.

18

Evaluation of Bioremediation in A Coal-Coking Waste Lagoon M.E. Leavitt,

D.A. Graves, Ph.D., and C.A. Lang, International Technology Corporation

A remedial investigation/feasibility study is being completed for a former coal

coking plant under the Superfund program. Bioremediation is one of the many

alternatives being evaluated as remedial tools for the waste lagoons. The

lagoons contain elevated concentrations of coal, metals, cyanide, phenolics and

polyaromatic hydrocarbons. A bench and pilot-scale study was designed and

executed to determine the overall effect of bioremediation on the organic contam-

inants and to collect the data to determine the overall feasibility of implementing

this technology in a full-scale system.

The bench-scale testing included a respirometer study using 10:1 and 50:1

ground water:soil slurries. The results indicated that an abundant population of

'.'iVble organisms were capable of degrading the organic carbon present in the

soil and water. The biologically-active slurries exhibited greater that 99% reduc-

tion in the specific organic contaminants.

The pilot study included: a) an in situ subsurface remediation system using a

center injection well and perimeter recovery wells, b) sprinkler nutrient injection

system to treat the unsaturated zone. Injection water was supplemented with

nutrients and hydrogen peroxide (for the subsurface treatment) for six months of

continual operation. Physical, biological and contaminant data were collected on

a weekly and monthly basis.

The physical and chemical data indicated that oxygen and nutrients were

being effectively transported throughout each system. Microbial populations

modestly increased, and on occasion have decreased. Contaminant data indi-

cated that total petroleum hydrocarbons, polyaromatic hydrocarbons and pheno-

lics have declined, however, significant variability exists.

The pilot study is scheduled to continue for an additional twelve months, and

the data will be used to determine the final record of decision for remediation of

these lagoons.

19

Evaluation Process For The Selection of Bioremediation Technologies For

Exxon Valdez Oil Spill Val J. Kelmeckis, NETAC

In the early cleanup stages of the Valdez spill, EPA and EXXON recom-mended that two bioremediation products should be applied to supplement

other more traditional cleaning methods. These two products are fertilizers that

enhance the action of naturally occurring microorganisms. Several weeks after

application, the beaches where they were used appeared cleaner than control

beaches nearby. However, the results were difficult to verify scientifically in the

field.

Soon after the spill, pressure began to build to allow other private sectorbioremediation companies to use their products in Alaska. The EPA and Coast

Guard had received many proposals.

NETACIn November 1989, EPA asked the National Environmental Technology Appli-

cations Corporation (NETAC) to establish criteria by which bioremediation

products for cleaning up oil spills could be judged. The criteria were to be

practical and consider oil spills in general as well as the Exxon Valdez Spill.

NETAC assembled an independent panel of expert scientists from industry,

academia, and applied research organizations. The panel developed a set of

criteria for evaluation how bioremediation technologies for oil spill cleanup couldbe chosen.

Next, EPA requested proposals, through an announcement in the Commerce

Business Dail outlining proposal criteria developed by the NETAC panel, for bio-remediation products that could be used to clean the contaminated beaches.

Thirty-nine world-wide proposals were submitted for review.

NETAC reconvened the panel in March 1990 to review the proposals. Ten

bioremediation technologies were recommended to EPA for laboratory testing.

The purpose of this laboratory evaluation was to further qualify the technologies

for possible use on the remnants of the EXXON Valdez oil spill in Alaska during

the Summer of 1990.

20

In June 1990, the NETAC panel reviewed the results of the laboratory tests on

the ten technologies and recommended two to EPA for field testing on the weath-ered crude oil in Prince William Sound. Both products contain naturally occurringmicrobial cultures with the addition of fertilizer. A field test protocol developed bythe EPA and reviewed by NETAC's expert panel was submitted to the agenciesresponsible for cleanup of the Alaskan spill. Field tests of the two products has

been completed with results of the test expected by the end of 1990.

NETAC has established criteria that leave the agencies better prepared toreview future bioremediation proposals. EPA plans to institutionalize the lessons

learned under this process for future marine oil spills. Protocols have beenestablished for alternative bioremediation technologies to be considered in the

future.

Full-scale Bioremediation of Contaminated Soil and Water Geoffrey

Compeau, Ph.D. and William D. Mahaffey, Ph.D., ECOVA Corporation and LoriPatras, Unocal Corporation

Biological processes have been used on a large scale to remediate petroleumhydrocarbons, pesticides, chlorinated solvents, and halogenated aromatic hydro-

carbons. Biological treatment of contaminated soils may involve solid-phase,slurry-phase, or in situ treatment techniques. This paper will review the general

principles of bioremediation and discuss the application of these techniques for

full scale cleanup.

Up to 280,000 cubic yards of soil on the site of a former oil refinery tank farmis contaminated with up to 15,000 parts per million (ppm) of petroleum hydrocar-bons. The site posed significant challenges due to its size as well as depth andrange of contamination. The implementation of biological remediation required

the design of a Land Treatment Unit (LTU) and a remedial program which would

support the treatment of a significant amount of contaminated soil within a restric-tive time schedule. Once this scenario was developed, the LTU was prepared fortreatment and excavation and placement of soils began. Currently, the LTU area

encompasses 27 acres of a 45 acre site.

21

A mobile laboratory has been placed onsite and is staffed with chemists andmicrobiologists who analyze up to 100 so!' samples per day. This lab has been

designed and equipped to provide the necessary chemical and bioiogicalanalysis to fully support the excavation and bioremediation program. Onsite bio-logical treatment activities include irrigating, aerating, and tilling the soil to bringmicroorganisms, contaminants, and oxygen into contact with each other topromote biological degradation. Chemcal and microbioiogical monitoring con-

ducted throughout the remediation process ensures that treatment levels arebeing met. In addition, numerous site studies were conducted in attempts todefine and enhance the r-mediation process.

A solid-phase bioremediation program was successfully completed at a

former wood treating facility; the objective was to biotreat approximately 17,000cubic yards of soil contaminated with petroleum products, pentachlorophenol,

and poly nuclear aromatic compounds.

The bioremediation program consisted of the construction of an onsite soiltreatment facility, operation of the facility for approximately six months, and siterestoration. Due to the treatment area size, the contaminated soil required treat-ment in tvvc, !vers or lifts. Baseline, verification, and clearance sampling and

analysis was conducLed for eac', of the two lifts by samples collected anddelivered to the ECOVA project laboratory. Treatment operations consisted ofdaily aeration of the upper lift soils with the addition of water and nutrients topromote and maintain optimum microbial activity anc maintain critical remediatior

parameters. Th:is data and the logistics of this full-scale remediation will becompared to another site containing PCP in soils that was approachied through aremediation Fxneme involving soil washing and aqueous-phase biotreatment.

22

4.1.3 SESSION Ill-TECHNICAL ISSUES AND CONCERNS IN ENVIRON-MENTAL BIOTECHNOLOGY IMPLEMENTATION

Feasibility and Other Considerations for Use of Bioremediation in Subsur-

face Areas Karolyn L. Hardaway, Ph.D., Texas Eastman Company

Texas Eastman Company, a manufacturer of chemicals and plastics, is using

a pump and treat system to remediate hydrocarbons at a site. In situ bioremedi-

ation of the subsurface area is being investigated as a more effective technology

to clean the site and to reduce potential environmental impacts. To verify the

efficacy of the tecnnology for this application, two laboratory studies were per-

formed. In the first study, laboratory analyses cnaracterized the groundwater and

soils from field borings to define factors that would enhance, complicate, or

preclude in situ biodegradation. Most data were favorable for bioremediation.

Low numbers of microbes in the saturated soils were offset by the large popula-

tion of microbes contained in the groundwater. Necessary oxygen for aerobic

biodegradation could be supplied using hydrogen peroxide since it was not

completely oxidized by soil or water samples. Results of nutrient addition and

hydrogeological evaluation indicated that soil characteristics in most areas were

amenable to in situ biodegradation. The second phase, a bench-scale treatability

study, was completed to establish criteria for a bioremediation field trial and

determine degradation rates. The criteria developed for the field trial is currently

bE ing evaluated. This treatability study indicated that in situ bioremediation in

.ther aerobic or denitrifying atmosphere is a viable method of reducing the

hydrocarbon content of the site.

Integration of Biotechnology to Waste Minimization Programs Godfred E.Tong, Ph.D., Monsanto Co.

Chemical engineers need to view biotechnology as one of three treatment

technologies availahle for use in waste minimization projects. As basic under-

standing on the behavior of industrial biological catalysts and bioreactors

23

emerge, biotechnology needs to be evaluated along with physical and chemical

technologies to determine its role in an integrated approach to the design Ui

cost-effective and reliable waste minimization systems.

This presentation provides four different levels for the evaluation of the poten-

tial role of biotechnology in waste minimization projects. These four levels are as

follows:

1. Biotechnology as a treatment alternative to physical and/or chemical treatment.

2. Biotechnology as a complementary too: either pre or post-treatment to physio-

chemical treatment technolagies.

3. Biotechnology as a model to arrive at "biomimic" non-living systems approach.

4. Biotechnology as a source of analytical monitoring tool of pollution problems.

Industrial examples for each of the potential roles of biotechnology will be

made in this presentation.

Bio-Remediation of Explosives Contaminated Soils (Scientific Questions/En-

gineering Realities) Craig A. Myler, Ph. D. and Wayne Sisk, U.S. Army Toxic

and Hazardous Materials Agency

The explosives trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine

(RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) have found almost exclu-

sive use as military compounds. As such, the liability for the wastes from past

production and processing of these compounds resides, in almost all cases, with

the federal government. As the total volume of soils contaminated with these

wastes may be substantial, and current remediation costs are high, considerable

effort is underway to develop cost effective alternatives for clean-up. Biological

methods offer the greatest potential for achieving this goal in the near term.

While extensive research has been conducted on the metabolism and environ-

mental fate of these c&mpounds, little information is available for the design of

systems capable of exploiting the natural process of utilization. Seeming conflicts

between laboratory data and pilot and field scale studies makes implementation

difficult. A discussion is presented on the current state of the art in explosives

24

contaminated soil remediation by biological means. Solid phase treatment bycomposting, aqueous phase treatment by slurry reactor, and inoculation usingprepared cultures will be presented. Problems in fielding these methods will alsobe described. These include methods and frequency of sampling and analysis,final product disposition and fate of the explosives, safety and handling con-

straints and the costs associated with implementation.

Practices, Potential, and Pitfalls in the Application of Biotechnology to Envi-

ronmental Problems Carol D. Litchfield, Ph.D., Environment America, Inc.

The use of microorganisms to remediate wastes is not a new technology. Infact, it predates not just modern biotechnology but even the recognition of micro-

organisms. Composting, and even waste water treatment plants, have been

around for a very lot g time. What is new is the recognition that the conceptsembodied in these "ancient" techniques can be applied to the cleaning up ofmodern contaminated groundwaters, soils, and even process streams. In therush to embrace this "new" technology, many have forgotten that there have

been limitations to composting and waste water treatment systems, and many ofthese same limitations will apply in the newer applications. Nevertheless, it is still

appropriate to examine how biotechnology is being utilized today to solve someof the current environmental problems such as groundwater and aquifer contam-ination, cyanide leachate from ore extractions, and landfill leachate. We will also

examine some of the exciting potentials for the application of biotechnology tosuch developing areas as waste minimization and agricultural pest control, whileremaining mindful of currently known or perceived limitations to the application of

biotechnology to solving environmental problems.

What is the Km of Disappearase? Ronald Unterman, Ph.D., Envirogen, Inc.

It is critically important that throughout biodegradation process developmentresearch (and ultimately commercial marketing), investigators clearly demon-

strate that their bacterial soil decontamination results are unequivocally due tobiological activity. Too often, the results of some studies have not been able to

quantitatively account for the disappearance of the target substrate. In some

25

cases, high!y hydrophobic contaminants are redistributed in reactors or sorbedto unsampled locations in these reactors. In other studies with, for example,volatile organics, vigorous aeration has potentially volatilized the target instead ofbiodegrading it. Therefore, biodegradation studies and demonstrations must bedesigned to determine as best as possible a mass balance of the target sub-strate and hopefully demonstrate the products of this transformation (ideally,carbon dioxide and water).

One such pitfall that can be encountered in these biodegradation studies canbe illustrated by research that the author and his co-workers have been conduct-ing on the biodegradation of PCBs. It is possible that an observed PCB congenerdepletion in a "biodegradation" process is actually due to physical loss of thePCB and not the true biological degradation. With Aroclor studies, these pro-cesses can easily be distinguished because biodegradation results in depletionof specific congeners yielding GC profiles that are distinctly different from thoseof the original Aroclor mixtures. Physical depletion, on the other hand, results inuniform depletion of all congeners (e.g., adsorptive loss) or depletion of lowerchlorinated congeners due to their higher volatility (e.g., evaporative loss). Theproduction of PCB metabolites is, of course, another unequivocal method fordemonstrating the biological basis of PCB depletion.

In an attempt to distinguish these different depletion mechanisms and to dem-onstrate this pitfall, we established a mock, nonbiological "biodegradation"process. PCB-contaminated soil was incubated with stirring in the absence of

bacterial inoculum and with a constant stream of inert gas (argon). Following 19days of incubation, this reactor was analyzed for PCBs in all locations. Althoughthe samples taken from the middle of the soil slurry throughout the 19 days didshow greater than 90% depletion of PCBs, the final mass balance was able toaccount for all of the PCB in the reactor by analyzing the physical components ofthe reactor (glass walls, stirrer, etc.) as well as coalesced PCB droplets in thebottom of the reactor.

Other studies with volatile organic compounds (e.g., TOE) have required thatthis research be done in sealed systems. A complete mass balance was there-fore determined relative to sterile controls. Such studies, when done under these

26

conditions, do clearly demonstrate that compounds such as TCE can bebiodegraded and not volatilized. However, in the final configuration of full-scalebioreactors, the control of aeration will be a critical parameter, as well as monitor-ing of the bioreactor offgas.

In summary, it is imperative that for bioremediation to be accepted as a safe,cost-effective technology, researchers must unequivocally demonstrate that theloss of the target substrate is indeed due to biological activity and not someother, non-biodegradative effect.

Use of Treatability Studies in Developing Rernediation Strategies for Con-taminated Soils Michael J. McFarland, Ph.D., Ronald C. Sims, Ph.D., andDivision of Environmental Engineering, Utah State University and James W.Blackburn, Ph.D., Energy, Environment and Resource Center, The University ofTennessee, Knoxville

Misunderstanding and confusion regarding use and interpretation of treatabil-ity study data has led to difficulty in relating treatability study results to actual per-formance in field applications. Much of the difficulty stems from misconceptionsconcerning the objectives of treatability studies, inadequate experimental design,and misapplications of treatability study results for subsurface remediation.

Treatability studies are used to provide specific information concerningchemical mass balances for a waste/soil mixture. By applying the chemical massbalance in treatability studies, the distribution of contaminants among subsurfacephases (e.g., gas, water, oil, and soil) can be characterized and used as a basisfor treatment technology evaluation and selection. They may also be used toevaluate potential application of treatment technologies at field scale by evaluat-ing and comparing rate and extent of remediation among several technologies.Recognition of uncertainties in the data, for example the use of confidence limitson degradation rates and partition coefficients, represents important informationfor making decisions at field scale.

27

Information obtained from treatability studies, conducted as mass balance

studies including laboratory screening, bench and pilot-scale studies, can be

combined with information concerning site and waste characteristics in order to

determine applications and limitations of each potential remediation technology.

Results of treatability studies and site characterization data can be used in simu-

latior (e.g., -,dthemalicai modeb;ng) in order to: (1) determine containirert

requirements to prevent contamination of off-site receiver systems; (2) develop

techniques to maximize mass transfer of chemicals affecting microorganisms

activity; and (3) design a cost-effective and efficient monitoring program to

evaluate effectiveness of treatment at field scale.

Biodegradation of Mixed Solvents by a Strain of Pseudomonas J.C. Spain,

Ph.D., Air Force Engineering and Services Laboratory

Mixtures of aromatic solvents are poorly degraded by bacteria

because most strains can attack only a few closely related chemicals. In

addition, simultaneous degradation of alkyl- and chloro-substituted com-

pounds is precluded by misrouting of intermediates and the resulting inhi-

bition of enzymes. The use of mixed cultures can overcome such

problems but mixed cultures respond very slowly to changes in substrate

feed. Pseudomonas sp. strain JS1 50 grows on a wide range of substituted

aromatic compounds including: benzene, toluene, chlorobenzenes,

phenols, and naphthalenes. It also degrades alkyl- and chloro-substituted

compounds simultaneously via a modified "ortho" ring-fission pathway.

Bench-scale experiments in stirred bioreactors indicate that strain JS1 50

completely degrades complex mixtures of aromatic solvents added

directly to the bioreactors without prior dilution. Chlorobenzene or dichlo-

robenzene served as the most effective inducers and toluene was the least

effective. Cells induced with chlorobenzene cometabolized several

compounds including TCE, chlorophenols, and cresols. Absence of

metabolites in the culture fluid suggested that these compounds were min-

eralized even though they could not serve as inducers or as sole sources

of carbon. The results indicate that pure cultures of bacteria can be useful

for biodegradation of complex mixtures of hazardous wastes.

28

4.1.4 SESSION IV-NONTECHNICAL ISSUES AND CONCERNS IN ENVI-RONMENTAL BIOTECHNOLOGY IMPLEMENTATION

The Field Implementation of Bioremediation: An EPA Perspective Fran

Kremer, Ph.D., Office of Research and Development, U.S. Environmental Protec-

tion Agency

This past February, the EPA Administrator called a meeting with over 70 rep-

resentatives from biotreatment companies, site cleanup contractors, industry,

academia, environmental organizations, EPA and other Federal agencies. This

meeting was designed to identify strategies to increase the use of bioremediation

for the cleanup of hazardous wastes.

One of the major themes highlighted in that meeting was the need to expandour field experience using this technology. Even though bioremediation is a

viable technology to treat some hazardous wastes, it has not been fully utilized

for the many different types of sites requiring remediation. Solid performance

data is needed to document the capabilities of this technology. It was recom-mended that the Agency serve as a focal point in fostering field tests, demonstra-

tions and evaluations of bioremediation, using good test protocols anddocumentation of results.

Based on this recommendation, the Office of Solid Waste and EmergencyResponse (OSWER) and the Office of Research and Development (ORD) have

instituted a Bioremediation Field Initiative using sites where bioremediation is

planned or in progress. This initiative provides assistance to the Regions and

states in conducting field tests and carrying out evaluations of site cleanups

using bioremediation. Sites considered in this field initiative include CERCLA.,

RCRA corrective action facilities and UST sites. This initiative is designed to 1)

more fully document performance of full-scale field applications of bioremedi-

ation, 2) provide technical assistance for sites in a feasibility or design stage tofacilitate the conduct of treatability studies, field pilot studies, etc., and 3) regularlyprovide information on treatability studies, design and full-scale operations of bio-

29

remediation projects. This Program is intended to provide information on the

operation of biological treatment systems for a variety of wastes and

contaminated matrices and provide current cost and performance data.

A Historical Perspective: Does Good Press and/Or Good Science Generate

Demand? Thomas G. Zitrides, Bioscience Management, Inc.

Public perception of microorganisms, after Pasteur, has involved a dichotomy

between harmful and beneficial "germs". On the one hand, various commonphobias, e.g., the Andromeda Strain; on the other, especially in the environmen-

tal engineering community, blind reliance that naturally-occuring microorganisms

will "just be there" to dispose of waste products, e.g., to treat sewage and

industrial wastes (of the "ubiquity principle"). As a result, both regulators and

environmental professionals have been slow to accept bioremediation, either withor without exogenous naturally-occuring microbes. These perceptions are

reflected repeatedly in the press, in spite of the documented effectiveness of bio-

remediation techniques.

Regulators are influenced by public phobias as exemplified by the initial ruling

out of the use of "non-indigenous, naturally-occuring" microbes to speed degra-

dation of shoreline deposits after the Valdez spill. Compounding the problem are

misconceptions of genetic engineering and its confusion with commonly-used

accepted techniques of selective adaptation of microbial consortia. The media

have both fanned public fears and helped overall bioremediation. Sensational-

ism, the lack of follow-up, lack of technical competence, and the fairness doctrine

which involved obtaining a contrary opinion on virtually any report, have

contributed to the problem.

We are now at the peak of the typical 5-year cycle of press interest in biore-

mediation. A concerted educational effort is required by the bioremediationindustry to maintain interest and acceptance. Neither regulators, the press, nor

the general public have sufficient information at present to evaluate bioremedi-

ation programs realistically.

30

Ways to Identify and Obtain Rights to Technology From Federal Facilities

John C. Corey, Ph.D., Westinghouse/Department of Energy

The federal government is a storehouse of useful and valuable research infor-

mation. In the past, American industry has felt that obtaining access to this infor-

mation was both difficult and non-rewarding. Congress recognized this

legitimate concern of the citizens. To correct these shortcomings, Congress

passed a series of acts in the 1980's to facilitate the movement of technology to

the private sector including the Bayh-Dole Act of 1980, the Stevenson-Wydler Act

of 1980, the Technology Transfer Act of 1986 and the National Competitiveness

Technology Transfer Act of 1990. As improvements are identified in these laws,

additional acts will be forthcoming.

The importance of these acts to the biotechnology arena is that a major focus

in the 1990's at many of the federal agencies is environmental restoration and

waste management. The federal government will be utilizing major research

capabilities to identify superior methodologies to comply with the desires of our

citizens to enjoy a safe and clean enviropment. The technology transfer acts

encourage industry to access the unique capabilities at federal facilities. Some of

the methods include cooperative research and development agreements

between industry and government, personnel exchange, research and develop-

ment, contracts, patent licensing, small business innovation research program,

consulting by laboratory scientists, and technical documents and software.

To facilitate the interchange and to provide a focal point for entry to a specific

federal facility, all major research facilities have an Office of Research and Tech-

nology Applications (ORTA). The ORTA office acts as a clearinghouse for inqui-

ries, oversees patent licensing, facility use agreements and is the appropriate

place to access a particular facility. For more general inquiries there is the

Federal Laboratory Consortium (FLC). The FLC represents all federal agencies

with active research activities including the Department of Defense, Environmen-

tal Protection Agency, NASA, Department of Energy, National Institutes of Health,

Department of Agriculture, and others. The FLC is an excellent point to enter the

entire federal research establishment when specific knowledge of where the infor-

mation resides is lacking.

31

An Overview of Current Attitudes on the Use of Biotreatment For CleanupWilliam J. Lacy, Dr.Sc., P.E., D.E.E., Lacy & Company

This paper covers how EPA and industry are emphasizing biotechnology

solutions for the treatment of hazardous wastes. Also, to be mentioned are the

integrated research, development and demonstration program plans on biosys-tem technology currently proposed by EPA.

Also, the Hazardous Waste Treatment Council's report which faults EPA's

1988 decisions for the use of bioremediation will be touched upon.

The projections based on the current market for biological treatment of haz-ardous waste is estimated, primarily on industrial sites and for industrial waste

waters.

Mentioned in the paper are a few of the significant institutional barriers tobiotechnology and the need to overcome these obstacles as well as the need for

the U.S. to either take the lead in the near term or increase our imports from

foreign competitors.

Views of a Project Manager: What are the Critical Facts Necessary to Win

the Confidence of the State Regulator? Frank R. Peduto, P.E., New York StateDepartment of Environmental Conservation, Bureau of Spill Prevention and

Response

Bioremediation has been among the few technologies which have demon-strated the ability to achieve regulatory levels. While this technology has demon-

strated a good deal of success, it is not without its drawbacks. Among these arehigh up-front costs for design, installation and costly nutrient supply throughout

the extent of the project. In order to justify this investment, there must be reason-

able assurances that it will work. The length of the project and the degree of

treatment are typically the least specified items. The regulator needs to be

assured of the degree of treatment which will occur and how long will it take. Theregulator must be satisfied that all potential negative impacts of this process have

been accounted for. They must understand and appreciate the limitations of the

process.

32

This paper will analyze a bioremediation project from two points of view: thatof the regulator who has an interest in achieving an environmentally soundsolution; and that of a contractor representing a responsible party who in turn

have a significant liability and economic interest.

It will discuss the type of information the contractor should be able to providethe regulator to satisfy his/her concerns and to assure a successful completion ofthe project. It will also identify what issues the regulator must be prepared toprovide the contractor in order that a proper estimate and design can beachieved. The establishment of project specifications will assure both parties ofwhat is expected of each other and will most accurately predict the final outcome

of the project.

Is There a Need for a Bioremediation Specialist Certification? Morris Levin,Ph.D., University of Maryland, Center for Public Issues in Biotechnology

The issues associated with waste disposal have been identified and describedin many places by observers from many perspectives. Legal, safety, technical,environmental, ethical, economic, and socio-political issues have been describedin depth over the past decades as public awareness increased of the magnitudeof the waste disposal problem. Each waste site or new treatment type raisessome or all of the same problems.

As the science and technology associated with waste disposal develops, thetechnical skills required to select and apply the appropriate remedial actionbecome more critical to the success of the project. In addition, knowledge ofapplicable statutes, both federal and local, community issues and the potentialfor long term environmental and public health problems of both the waste andthe treatment method is required before appropriate disposal can be achieved.

At present, private companies or municipalities operate waste disposal sitesand develop waste disposal procedures and regulatory agencies review andmonitor most, but not all, waste disposal practices. Both generally rely oninhouse staff or consultants for technical expertise. Often, however, the expertise

33

is not selected based on objective criteria but on availability of personnel, result-

ing in costly errors and delays. The result is increased cost, higher probability of

environmental damage, and greater potential for adverse health effects.

Many professional groups--ranging from pathologists to midwives to

ecologists--have instituted or considered instituting a certification or accreditationprogram which will assure that a supply of competent specialists will develop. As

will be discussed, the basis for these considerations is a recognized need for

public confidence in a profession or industry, an understanding that the area of

endeavor is becoming highly complex and thus requiring special skills or training,

and an interest in minimizing liability for the practitioner.

Many of these arguments are valid for biotechnology as it applies to bioreme-

diation. This paper will examine some of the issues involved in and define the

potential need for and value of a certification or accreditation program to the

bioremediation industry at the individual and corporate level.

Biological Monitoring Related to Demonstration of Waste Remediation C.W.

Gehrs, Ph.D., J.M. Loar, Ph.D., A.J. Stewart, Ph.D., J.F. McCarthy, Ph.D., L.R.Shugart, Ph.D., and S.M. Adams, Ph.D., Environmental Sciences Division, OakRidge National Laboratory

Techniques developed through various subfields of environmental biotechnol-

ogy will play major roles in remediation of waste sites at various Department of

Energy facilities. One such component is associated with biological monitoring.

This presentation focuses on the experience at Oak Ridge Reservation during the

past five years, drawing on specific examples of biological monitoring that have

demonstrated the efficacy of remediation activities, aided in identification and

prioritization of waste sites, or developed new biochemical techniques for assess-

ing the status of the environment and developing exposure assessments for

health risk assessments. Three components will be discussed: environmental

toxicology, in-stream monitoring, and biological markers.

34

Environmental toxicology can serve to identify and prioritize contaminants

requiring removal or treatment. It consists of a standard set of biological systemswhich provide real-time analytical evaluation often more sensitive than chemical

analyses. The use of in-stream monitoring to assess the status of the environ-ment, in which the wastes are found or released, determine the potential

exposure pathways (for man), identify potential treatment strategies and evaluateis a subfield of growing importance to developing waste management strategies.

Biomarkers, for example, refers to any of a series of biochemical or molecularindicators, that can be measured in plants and/or animals on waste sites or

through laboratory experiments with wastes treated through various scenarios.The organisms integrate temporally and spatially real world exposures to contam-inants developing exposure assessments for human health risks assessments aswell as data for environmental risk assessment.

The submitted manuscript has been authored by a contractor of the U.S.

Government under Contract No. DEAC05-840R21400. Accordingly, the U.S. Gov-ernment retains a nonexclusive royalty-free licence to publish or reproduce the

published form of this contribution, or allow others to do so, for the U.S.

Government purposes.

Federal Regulations: How they Impact Research and Commercialization ofBiological Treatment Sue Markland Day, Waste Management Research and

Education Institute, Center for Environmental Biotechnology, The University ofTennessee, Knoxville

Biology-based waste treatment is a creature of this nation's environmentalprotection laws. Without the Superfund; water, solid and hazardous waste man-

agement programs; and the requirement for leaking underground storage tankcleanup, little research would be underway and little commercial demand wouldexist for bioremediation. The choice of specific treatment technologies is, to alarge part, controlled by laws, regulations, and policies. Such governmental doc-

uments may encourage the uses of treatment or may regulate the treatment

methodologies (place constraints on the uses of the technology). Often these two

conflicting purposes are contained in the same statute.

35

This speech will introduce the audience to important federal environmentalregulations and actions, analyzing each for their positive and negative impacts ofbioremediation and new market creation specific to biology-based waste man-agement. After a presentation of specific key federal legislative and regulatoryrequirements important to bioremediation, the talk will conclude with an analysis

of future regulatory trends as they impact environmental biotechnology research

and commercial applications.

Six factors, which will be discussed in the speech, promise to contribute to theevolving biological treatment regulatory structure:

(1) The continued debate over the use of technology standards versus the use

of health or risk-based standards as treatment objectives.

(2) The high cost of treatment technologies, such as incineration, compared tothe anticipated lower cost of bioremediation.

(3) The "Not In My Backyard" syndrome which is encouraging on-site hazardouswaste treatment.

(4) An increase in funding for environmental biotechnology research.

(5) An anticipated rapid transition fro;n basic research on environmental biotech-nology to in-the-field applications.

(6) The public debate over safety issues associated with the plannedrelease of genetically-altered microbes.

36

4.1.5 SESSION V--INTERNATIONAL ACTIVITIES IN ENVIRONMENTAL BIO-TECHNOLOGY

Polluted Ieterogeneous Environments: Macro-scale Fluxes, Micro-scale

Mechanisms and Molecular-scale Control Geoffrey Hamer Ph.D., Institute for

Aquatic Sciences and Water Pollution, Swiss Federal Institute of Technology,

Zurich, Switzerland

Until some 10 years ago, microbiological research was dominated by an over-

emphasis on both pure monocultures and single carbon energy substrates, on

the one hand, and by the assumptions that most cultures are essentially

homogeneous and that any micro-scale heterogeneities were largely irrelevant to

culture performance, on the other hand. Whilst clearly, many important discover-

ies were made within the scope of these limitations, today's pressing problems

for effective, efficient and economic microbially mediated remediation

technologies for dangerously polluted environments require an entirely different

approach. The major question is how can microbial consortia be harnessed for

accelerated rate bioremediated processes in markedly heterogeneous, seriously

perturbed natural and/or inappropriately designed, man-made environments?

The way forward requires a firm factual and quantitative basis for describing the

microbial potential in real environments, but this will not be achieved by either

sequential monitoring or testing programmes. It requires imaginative conceptual

thinking followed by validation of the proposed concepts. The presentation will

evauate the present status with respect to such an approach, considering the

implications of both micro-scale mechanisms and molecular-scale control on

macro-scale pollutant flux mediation.

37

The Pilot Plant Testing of the Continuous Extraction of Radionucleides usingImmobilized Biomass Marios Tsezos, Ph.D., Department of Chemical Engineer-

ing, McMaster University, Hamilton, Ontario, Canada

The selective sequestering of metal ions from aqueous solutions by microbialbiomass has been termed biosorption, Biosorption of metal ions is a phenome-non exhibited by both alive and dead microbial cells. The detailed investigation

of the mechanism of biosorption has revealed that biosorption is aphysical-chemical process whereby selected areas of the microbial cell exhibithigh selectivity and specificity for the extraction and retention of specific metalions from aquatic solutions.1 This property is exhibited equally well or often better

by dead cells than by the same cells alive. The use of proper chemical solutions(eluants) is capable of reversing the equilibrium of biosorption bringing the bio-sorbed metal ions back in solution and freeing the biomass active sites for sub-

sequent reuse. 1

Complex solution ionic matrices present a special challenge in so far ascertain ions may compete with others during biosorption thus reducing thebiornass metal uptake capacity.2 Understanding of the mechanism by whichcoions can affect biosorption and the manipulation of corresponding solutionparameters, like pH, can offset adverse coion effects. 1,2,3,4

The small particle size of the microbial cell possesses however significantdifficulties in the full scale industrial application of biosorption as a metal extrac-tion and recovery process. The immobilization of the microbial jiomass into par-ticles of specified desirable physical and chemical properties alleviates thisdifficulty. Such a proprietary biomass immobilization process has beendeveloped by the author. The produced immobilized biomass particles havebeen shown to have custom made particle size, high porosity and favorable bio-sorptive equilibrium and kinetic properties. A mathematical mass transfer kinetic

1 Tsezos M., Biotech. and Bioeng. 29, pp 973-981, 19842 Tsezos M., Baird M.H.I., Shemilt L.W., The Chem. Eng. Journal, 32, pp 829- 838, 19863 Tsezos M., Nob S. H., Baird M.H.I., Biotech. & Bioeng., 32, 545, 19884 Tsezos M., McCready R.G.L., Bell J.P., Biote-1h. & Bioeng., 34, 10 1989

38

model of biosorption has revealed the most significant engineering parametersthat affect the immobilized biomass particle biosorptive behavior and hasresulted in improving substantially the behavior of the produced biosorbent parti-

cles.3

Over the last 15 years, the detailed study of uranium biosorption has led to thedesign, construction and operation of two pilot plants for the continuous biosorp-

tive recovery of uranium from the industrial biological leachates produced by

Denison Mines in the Elliot Lake area of Canada4 . The results from the two pilotplants have shown that the continuous biosorptive extraction and recovery ofuranium is possible. The feed to the pilot plant has an incoming uranium concen-

tration of 100 to 300 mg/L, a pH of 1.5 to 2.0 and a wide spectrum of cations and

anions such as sulfates, calcium, iron, aluminum, rare earths etc. Some of thesecations are present in concentrations as high as several thousand mg/L. Thepilot plant produces a uranium concentrate stream with uranium concentrations

as high as 14,000 mg/L.

An initial drop in the immobilized biomass uranium uptake capacity has beenstudied using techniques such as microprobe spectral analysis with digital image

analysis and infrared spectroscopy. Recent results from the above work have

suggested that the aluminum present in the biological leachate accumulates inthe microbial biomass cell wall in the form of amorphous aluminum-silica hydroly-sis products, resulting in a loss of uranium capacity. On the basis of the under-

standing of the mechanism of competition we have developed countermeasure

strategies, the first of which has been tested with success.

The paper will present an overview of the above advances made a McMasterin the area of the biosorption fundamental and the development of the corre-

sponding technology. This novel technology is a good example of the applica-tions of biotechnology in the areas of metal recovery and environmental pollution

control.

39

Research and Development Programs for Biological Hazardous Waste Treat-ment in the Netherlands Esther Soczo and Klaas Visscher, National Institute ofPublic Health and Environmental Protection, The Netherlands

Biotechnology is increasingly being applied in solving environme ital problems

in the Netherlands in the last decade. Micro-organisms have, of course, beenused for centuries to process organic waste produced by man. Man has discov-

ered that even non-naturally occurring substances such as polycyclic aromatic

and chlorinated hydrocarbons can be degraded micro-biologically. The bigadvantage of biological hazardous waste treatment is that the pollutants arebroken down into substances which are part of the natural cycle. This

environmentally-friendly process can contribute to the recovery of our environ-

ment.

Biological treatment processes have been applied in the Netherlands in thecleaning of gases from industrial installations, in treating of waste water and in thecleanup of contaminated soil. The first biological filter for waste gas cleaning was

developed in 1978. Since that time more than 200 biological filters have been

installed. An other important Dutch development is the anaerobic waste watertreatment. The design is being sold around the world. About 130 plants havebeen built between 1980 and 1988 inclusive, 90 of them abroad. Soil treatment is

still a new area in the application of biotechnology. Landfarming methods andin-situ biorestoration have already been applied for the clean up of sites mainly

contaminated with oil compounds. Bioreactors are in the development stage; the

first pilot plants are being tested at this moment.

Environmental biotechnology is being promoted by the Dutch governmentwithin the framework of q number of subsidy programs. Altogether 23 million

guilders (approx. $12 million) have been provided for environmental biotechnol-ogy projects in the past five years through a number of technology programs.

40

4.2 POSTER ABSTRACTS

PCR and LUX Constructions Bruce M. Applegate and C.M.B. Werner, Center for

Environmental Biotechnology, The University of Tennessee, Knoxvi!!e

The lux transposon Tn 4431 has been used in constructing bioluminescentreporter strains. This technology has an important application in Environmental Bio-

technology as bioluminescent reporters can be created for the biodegradation of

polyaromatic hydrocarbons. The Polymerase Chain Reaction (PCR) was used in

mapping the insertion site of the transposon Tn 4431 into the lower catabolic

pathway of the naphthalene degrading plasmid pKA 1. Using primers for specific

genes in the lower pathway of NAH7, a highly characterized naphthalene degrading

plasmid whose catabolic genes show distinct homology with pKA 1, and primers forthe insertion sequences of Tn 4431 it was possible to determine the insertion site of

the transposon. This approach provided both the actual gene which was disrupted

and the location of the transposon relevant to the transcription initiation site of the

lower pathway.

Bioremediation Studies of a Coal Gasification Site Soil C. Baker and C. F. Kulpa,

Ph.D., Department of Biological Sciences and the Center for Bioengineering and

Pollution Control, University of Notre Dame

The bioremediation of wastes containing numerous and diverse components is

most practically achieved by utilizing a mixed population of microorganisms whose

members exhibit a variety of metabolic capabilities. The aim of this study is to

examine the metabolic interactions among the members of a mixed microbial pop-

ulation enriched from a chemically complex waste site. Samples of soil from a coal

gasification site were enriched with n-hexadecane or naphthalene, giving rise to two

mixed cultures whose members demonstrated different metabolic properties. The

ability of the n-hexadecane enriched culture to emulsify hydrocarbons was attributed

to the presence of a surfactant producing organism identified as Acinetobacter cal-

coaceticus. Other members of the hexaclecane enriched culture was comprised of at

least two distinct Pseudomonas species, and other isolates thought to bePseudomonas or Aeromonas species as well. Both cultures exhibited the ability to

utilize naphthalene, though only one isolate from each culture was able to utilize

41

naphthalene as a sole source of carbon in pure culture. To determine the effects ofmetabolites produced by the naphthalene utilizing organisms on the growth of thehexadecane enriched culture, growth was tested in minimal medium supplementedwith cell-free medium from a two-week-old culture of the naphthalene enrichmentwhich had been grown on 500 ppm naphthalene. Growth on this medium was onlyslight, but when hexadecane was included the culture grew readily, demonstratingno inhibitory effects of naphthalene breakdown products on the metabolism of hexa-decane. An emulsification assay was employed to quantitate surfactant activity inmicrobial consortia. The implications of exploiting the emulsifying ability of onebacterial strain to facilitate better utilization of sparingly soluble polyaromatic hydro-carbons (PAHs) is addressed.

Plasmid and Catabolic Gene Frequencies in Linear Alkylbenzene Contaminatedand Pristine Freshwater Ponds Alec Breen, Luis Jimenez, Ph.D., and GaryS.Sayler, Ph.D., Center for Environmental Biotechnology, The University of Tennes-see, Knoxville and Thomas W. Federle, Ph.D., Environmental Safety Department,Procter and Gamble Co., Ivorydale Technical Center

A pond receiving high levels of linear alkylbenzene sulfonate (LAS) and a pristinepond were compared in their abilities to mineralize LAS. Individual isolates fromthese sites were surveyed for the presence of plasmid DNA and specific genesinvolved aromatic hydrocarbon oxidation. In general both LAS impacted and controlsites demonstrated mineralization capabilities though the pristine site demonstrateda lag period relative to the contaminated site. Frequency of plasmid DNA was slightlyhigher at the control site (68%) than at the contaminated pond (44% in the weed bedand 51% in the pond water). Contaminated and control sites harbored similar fre-quencies of aromatic catabolic genes. These data suggest that the genetic potentialfor degradation of aromatic compounds, as assessed with these catabolic geneprobes, is roughly the same and that acclimation to LAS is a key factor for LAS bio-degradation. Enrichment cultures from the pond sites as well as activated sludgefailed to yield any pure cultures capable of complete LAS degradation. This datasupports the hypothesis that LAS mineralization is mediated by consortia rather thansingle organisms in environmental settings.

42

Biological Removal of Biochemical Oxygen Demand from a Boron Contam-inated Industrial Waste Stream Kandi Brown and Janet Nichols, IT Corporation

Biotechnology Applications Center

The objective of this investigation was to determine whether an industrialwaste stream contaminated with 462 parts per million (ppm) boron wasamenable to biodegradation to meet Publicly Owned Treatment Works(POTW) effluent criteria. The study employed a 5.0 liter (L) New Brunswick

Scientific Bioflow III fermentation unit modified to recycle solids. The systemwas maintained at a biological solids retention time (BSRT) of 12 days withhydraulic retention time (HRT) set points ranging from 1 to 4 days. The fer-mentation unit was inoculated with 5.0 L of activated sludge and maintained atthe following set points: 2 ppm dissolved oxygen, 7.0 - 8.0 pH, 500 revolutions

per minute (rpm) agitation, and 250 C temperature. The biochemical oxygen

demand (BOD 5) of the influent feed stream was reduced by 98 percent to aneffluent concentration of 15 ppm during operation at a 4 day HRT. The

percent reduction of the influent BOD 5 concentration during operation at a 1day HRT was 79 percent resulting in an effluent concentration of 202 ppm.The POTW effluent limit for BOD 5 was 550 ppm. Total organic carbon (TOC)and chemical oxygen demand (COD) reniuval efficiencies ranged from 98 to64 percent, respectively, throughout the study. In conclusion, bioremediationwas a viable technology to employ in the remediation of the influent wastestream. The study is currently moving to pilot scale.

Bioremediation of Hazardous Waste in a Slurry Reactor--The EIMCOBiolift Reactor Gunter Brox and Douglas E. Henify, EIMCO Process Equip-

ment Company

Bioremediation in the slurry phase offers distinct advantages over in-situtreatment, land treatment, or composting: Better control of environmental con-ditions, i.e., pH, temperature, aeration, nutrients, desorption of contaminantsinto aqueous phase, and thus, more rapid treatment of certain wastes. Aclosed reactor allows volatile emission control and operation in aerobic oranaerobic mode Gas recirculation of the off-gas back into the slurry by

43

means of the diffusers allows biodegradation of the organic volatiles. Opera-tion in a gas recirculation mode also allows oxygen enrichment and thusbetter oxygen transfer in the slurry. The addition of co-metabolites is possibleas well. Potentially, gentically engineered bacteria will first be used in closed

reactor systems.

The EIMCO Biofift ReactorTM is a modified slurry agitator that uses acentral airlift, bottom rakes, and an innovative diffuser design to achieve thebasic objectives of mixing and aerating a slurry to sustain aerobic biodegra-dation processes. It can handle slurries of 25 - 50 vt% solids concentrationsand provide mixing and aeration at a much lower energy consumption thanconventional liquids/solids contact reactors. Several continuously fed, com-pletely stirred reactors are often arranged in series to achieve optimum degra-

dation kinetics and to meet low clean-up standards.

The BioliftTM Reactor is being used in several RCRA and Superfund appli-cations. A wide mix of organic contaminants have been degraded in differentsoil and sludge matrices.

A brief overview of other bioslurry reactors will be given and their develop-mental status will be discussed.

In Situ Depletion of Pentachlorophenol (PCP) from Contaminated soil by Phan-erochaete spp. Diane M. Dietrich and Richard T. Lamar, Ph.D., Institute for Micro-b -l and Biochemical Technology, US Forest Service Forest Products Laboratory

The purpose of this field study was to determine the ability of two white-rot fungito deplete pentachlorophenol (PCP) from soil, that was contaminated with a com-mercial wood preservative. Inoculation of soil containing 250-400 ug g--1 PCP witheither Phanerochaete chrysosporium or P. sordida resulted in an overall decrease of88% to 91% of PCP in the soil in 6.5 wk. This decrease was achieved under subopti-mal temperatures for the growth and activity of these fungi, and without the additionof inorganic nutrients. Since this soil had a very low organic matter content, peat wasincluded as a source of organic carbon for fungal growth and activity. A small per-centage (8% to 13%) of the decrease in the amount of PCP was a result of fungal

44

methylation to pentachloroanisole (PCA). Gas chromatographic analysis of sample

extracts did not reveal the presence of extractable transformation products other

than PCA. Thus, if losses of PCP via mineralization and volatilization were negligible,

as they were in laboratory-scale studies, most of the PCP was converted to nonex-

tractable soil-bound products. The nature, stability, and toxicity of soil-bound trans-

formation products, under a variety of conditions, must be elucidated before use of

these fungi in soil remediation efforts can be considered a viable treatment

technology.

Biolumninescent Sensing Technology Paul Dunbar, Center for Environmental Bio-

technology, The University of Tennessee, Knoxville

Bacterial luminescence has shown to he a useful phenotype for laboratory

research and potential for field studies. The various methods of bioluminescence

measurements found in the literature include eyesight, 35 mm photographic film,

X-ray film, and photon sensitive electronic equipment. Photomultipliers have been

used primarily to monitor bioluminescence on-line in biological fermentors. Fiber

optic sensors (or optrodes) allow in situ measurements which minimally disturb

systems.

The combination of small optrodes and photomultipliers offer a potentially

powerful tool for remotely sensing the bioluminescence of bacterial strains incomplex environmental conditions. A photomultipler-optrode system has been con-

structed to measure the on-line light emission of bioluminescent strains in a simu-

lated groundwater system.

Isolation and Characterization of mRNAs Transcribed by Catabolic Genes from

Soil Sediment Microorganisms J.T. Fleming, Ph.D. and G.S. Sayler, Ph.D., Center

for Environmental Biotechnology, The University of Tennessee, Knoxville

DNA extracted directly from complex environmental samples may be used to

quantitatively determine the abundance of catabolic genes by nucleic acid hybridiza-

tion. We sought to compliment DNA hybridization analysis with the isolation of cata-

bolic mRNAs from solid microorganisms.

45

Sterile soil was inoculated with 108 and 109 cells/g soil from induced and unin-duced cultures of Pseudomonas putita (PpG7) and subsequently total RNA was

isolated by in situ lysis, phenol/choroform extraction and pelleting through CsCI.RNA samples electrophoresed on denaturing gels and stained with ethidium

bromide displayed undegraded 16s and 23s ribosomal subunits after DNase treat-

ment. Following transfer to nylon membranes, hybridization with 3 2P-labeled nah

ABCD probe and autoradiography, distinct mRNA bands unaffected by DNase

digestion were observed with induced but not uninduced RNA.

An uninoculated Manufactured Gas Plant (MGP) soil contaminated with polyaro-

matic hydrocarbons containing 2 X 105 nah positive cells/g and a creosote contam-inated soil containing 1 X 108 nah positive cells/g, both previously stored at 40 C,

were warmed to room temperature for 2h in a water slurry and RNA was isolated asabove. 10 ng/g of nah mRNA was obtained from the MGP soil as determined by

RNA slot blots using PpG7 mRNA to make a standard curve. Northern blots of

DNase treated creosote soil RNA probed with a 3 2P-labeled in vitro transcribed nah

ABCD RNA probe displayed discrete bands of similar mobility to PpG7 mRNA.

These experiments suggest that in situ RNA isolation may be used to quantitate the

expression of environmental catabolic genes.

Effect of Direct Surfactant Addition to Petroleum Biodegradation in Soil Under-going Laboratory Scale Land Treatment Duane Graves and Maureen Leavitt IT

Corporation, Biotechnology Applications Center

The direct application of surfactants to petroleum contaminated soil has beenproposed as a mechanism to increase the bioavailability of insoluble compounds.

Solubilization of hydrophobic compounds into the aqueous phase appears to be asignificant rate limiting factor in petroleum biodegradation in soil. Nonionic surfac-tants have been developed to solubilize a variety of compounds, thus increasing the

desorption of contaminants from the soil. In this study, laboratory treatability studies

which emulate land treatment scenarios were used to monitor the bioremediation of

petroleum contaminated soils. In efforts to achieve the lowest levels of residual

petroleum hydrocarbons in the soil following biotreatment, 0.5 and 1.0% (volu-

me/weight) surfactant was blended into soils under treatment. Two soil types were

46

studied, a high clay content soil and a sandy, silty soil. In both cases, the addition of

surfactant (Adsee 7990, a blend of ethoxylated fatty acids) stimulated biological

activity as indicated by increased heterotrophic colony forming units per gram of

soil. However, the increased activity was not correlated with removal of petroleum

hydrocarbons. In the sandy-silty soil, the amount of petroleum removed from the

treatment was actually less than that removed in the surfactant-free treatment. In the

clayey soil, no enhancement of petroleum hydrocarbon removal was observed in

surfactant-amended treatments. The results suggest that the application of surfac-

tants directly to the soil for the purpose of solubilizing hydrophobic compounds was

not successful in achieving greater levels of petroleum hydrocarbon removal.

A High-Solids High-Yield Methanogenic Reactor: Microbial Biomass, Commu-

nity Structure, Metabolic Status, and Activities David B. Hedrick, James B.

Guckert, Ph.D., Institute for Applied Microbiology, The University of Tennessee,

Knoxville, Brian Richards and William Jewell, Ph.D., Department of Agricultural and

Biological Engineering, Cornell University, David C. White, M.D., Ph.D., Institute for

Applied Microbiology, Department of Microbiology, The University of Tennessee,

Knoxville, Environmental Sciences Divison, Oak Ridge National Laboratory

A novel semi-continuously fed anaerobic biomass digester operated at high

solids (25%), thermophilic temperature (55 degrees C), and high pH (7.8 - 8.1), was

found to be a very efficient system for the conversion of plant biomass to methane

fuel. At 7.5 Lkg -1 day1 , significantly higher than any reported for particulate feed-

stocks. The organic loading rate was 24 gVS kg-1 day- 1 at the highest productivity.

Stable operation was observed for over 70 days. Best performance was observed

with the less valuable, high C/N feedstocks. The biomass, community structure, and

metabolic status of the high-solids system were determined over a feeding cycle by

analysis of eubacterial and methanogenic membrane lipids. Microbial activities were

determined by radiotracer analysis. Measures of biomass and community structure

showed little variation over the feeding cycle, while measures of metabolic stress

peaked at the disturbance of feeding and during starvation. Microbial activities, such

as rate of acetate turnover and methane production rate, varied systematically over

47

the feeding cycle. The evidence showed this system had selected for a microbial

community capable of rapidly utilizing feedstock when provided, and surviving inter-

mlttent starvation. This high-solids anaerobic digester system is a viable candidate

for a national biomass-to-energy system. The methods of analytical microbial

ecology provide the insight required for transferring applications to production.

Treatability Studies To Develop Strategies For the Bioremediation of Tetrahy-

drofuran Contaminated Groundwater Ronald J. Hicks, Ph.D., Bioremediation

Services, Groundwater Technology, Inc.

Industrial solvents, such as tetrahydrofuran (THF), are often found as contam-

inants in groundwaters due to accidental release or past disposal practices. Biore-

mediation represents a viable technology for the remediation of THF contaminated

groundwaters. Possible strategies for implementing bioremediation technology for

THF clean-up include the use of bioreactors, in-situ bioremediation, or a combination

of these alternatives. Groundwaters, contaminated with THF at concentrations of

between 200-1000 ppm, were collected and used in laboratory and pilot scale

studies to identify and optimize possible bioremediation strategies. Biodegradation

studies were performed to determine if indigenous bacteria could be effectively stim-

ulated to degrade THF. To simulate in-situ bioremediation, THF contaminated

groundwaters were amended with varying concentrations of nutrients and incubated

under aerobic conditions. Greater than 99% of the THF was degraded within 120

hours in nutrient amended samples while less than 3% was removed in either

unamended samples or poisoned controls. Bacteria capable of degrading THF

were isolated and acclimated to increasing THF concentrations. These cultures were

used as inocula in bench scale bioreactors to determine minimum retention times

necessary for degradation of 400 ppm feed stocks. Respirometer studies were per-

formed to optimize nutrient loading rates and for toxicity testing. Finally, engineering

evaluations were performed to determine the hydrological and physical limitations to

implementing in-situ bioremediation. The results of these studies were used to

design a closed-loop system comprised of a bioreactor for treating recovered

groundwaters coupled to a nutrient and oxygen infiltration system to stimulate indig-

enous THF degrading bacteria for in-situ treatment.

48

Mieralization of Linear Alkylbenzene Sulfonates by a Mixed Bacterial CultureLuis Jimenez, Ph.D., Alec Breen, Gary S. Sayler, Ph.D., The Center for Environmental

Biotechnology, The University of Tennessee, Knoxville and Thomas W. Federle,

Ph.D., Environmental Safety Department, Procter and Gamble Co., Ivorydale Techni-

cal Center

Although Linear Alkyl Benzene Sulfonates (LAS) are biodegradable that does not

mean that ultimate mineralization to C02 occurs. Previous studies have not reported

mineralization of LAS by pure or mixed bacterial cultures. A bacterial consortium

capable of LAS mineralization under aerobic conditions has been isolated from an

activated sludge chemostat. Samples from the chemostat were plated on YEPG

media for isolated colonies. Four different bacterial colonies were detected all Gram

negatives rod-shaped bacteria which grow in pairs and short chains. Bacteria have

shown morphological characteristics similar to the Pseudomonas spp. Based on the

methylene blue active substances assay primary biodegradation of LAS was faster

with the bacterial consortium than with the individual bacterial components. Bacterial

colonies were grown together in minimal medium and 14 C-LAS as the only carbon

source. After 13 days significant mineralization to 14CO2 was obtained. Individual

members of the bacterial consortium tested did not mineralize LAS. This study has

shown that the four bacteria complemented each other and synergistically brought

about a high rate of LAS mineralization suggesting a strong evidence for a degree of

metabolic cooperation between the four bacterial components.

Competition and Simultaneous Maintenance of Polycyclic Aromatic Hydrocar-bon and Chlorobiphenyl Degrading Bacteria in Continuous Culture Wade

Johnston, Center for Environmental Biotechnology, The University of Tennessee,

Knoxville

Continuous flow chemostat cultivation was used as a model system to investigatethe simultaneous removal of mixed contaminants by defined mixed microbial popula-

tions, and to evaluate the co-maintenance of wild type and engineered strainsinvolved in biodegradation. Experiments were conducted to determine the

interactions among severa! wild type polycyclic aromatic hydrocarbon (PAH) and

chlorobiophenyl degrading strains. A continuous culture system was constructed of

49

wild type and/or engineered lux reporter stains capable of degrading naphthalene or

4-chlorobiphenyl as sole carbon sources. Bacterial popu!ation dynamics were moni-

tored by growth on selective media. Naphthalene and 4-chlorobiphenyl degradingwild type strains showed simultaneous maintenance. At a dilution rate of 0.17 hr- 1,

the naphthalene and 4-chlorobiphenyl degrading strains were maintained at 107 and

2 x 106 cells/mi respectively for 268 generations. When the dilution rate was ra, ed

to 1.71 hr- 1 , the strains were maintained for an additional 604 generations.

A similar system containing the wild type naphthalene degrabng strain 5R plus

naphthalene degrading lux reporter strains was used. The wild type strain 5R, 5Rlux,

and Pseudomonas putida RB1 351 lux were maintained at 108, 1.3 x 103, and 5 x 104

cells/ml respectively at a dilution rate of 0.51 hr - 1 for 743 generations. At a dilution

rate of 1.71 hr- 1 , the 5R and P, putida RB1 351 lux strains were maintained as before

for an additional 992 generations but 5Rlux was nearly washed out.

A system containing an engineered lux reporter strain (Ps. putida RB1351lux) and

a community derived from a PAH contaminated soil was supplied with naphthalene

and phenanthrene as well as a soil extract. The lux reporter strain was monitored byspread plating on selective media and was maintained at 2.3 - 2.8 x 106 cell m1- 1 for

58 generations. Bioluminescence was maintained at 1.4 -2.3 x 10-8 amps. These

results demonstrate that the lux strain can successfully compete against a wild type

assemblage. The results demonstrate a useful chemostat approach for examininghigh order interactions among mixed populations promoting multiple contaminant

removal.

Benzene, Toluene and Xylene Biodegradation Under Denitrifying Conditions inSoil Columns D.S. Kosson, Ph.D., G.F. Bowlen, B. J. Stuart, and P.D. Taylor,

Department of Chemical & Biochemical Engineering, Rutgers University

A mixed microbial population was shown to simultaneously degrade toluene,

m-xylene and p-xylene under anoxic conditions with nitrate as the electron acceptor.

The columns were operated in an up-flow configuration, at an approximate velocity

of 10 cm/day (200 ml) and only BTX (benzene, toluene, m-xylene, o-xylene and

p-xylene) were simultaneously utilized as the carbon source. The soil column design

allowed multiport sampling, with off-gas quantification and analysis. The complete

50

transformation of up to 0.02 mM of each component was accompanied by produc-

tion of N2 and C02 and a concomitant drop in nitrate concentration. The toluene andm-xylene were removed before the first sampling port (14.3 cm) and the p-xylenewas removed by the upper sampling ports (58.7 cm and 73.0 cm, respectively).Removal of the nitrate effectively stopped the transformation of the hydrocarbons.

The physical forces, dispersion and adsorption to soil, affecting hydrocarbon flow

through the system were also investigated. The parameters affecting both dispersionand adsorption were fitted to a flow model by nonlinear regression. Dispersion withinthe flow system was quantified by ion tracer studies. Adsorption of the BX com-

p i.r,cs ' 'as quantified in batch serum bottle experiments, using multi-componentmixtures to determine the adsorption coefficients.

The complete transformation of benzene and o-xylene was not observed duringthe experiments. Incomplete transformation of toluene and m-xylene was observedat 0.2 mM of each component.

Molecular Analysis of Manufactured Gas Plant Soils for Naphthalene Mineral-ization J. Sanseverino, Ph.D., C.M.B. Werner, J. Fleming, Ph.D., B.M. Applegate,

J.M.H. King, Ph.D., G.S. Sayler, Ph.D. and J. Blackburn, Ph.D., Center forEnvironmental Biotechnology, The University of Tennessee, Knoxville

New molecular tools are being developed and tested to ascertain the biodegrad-ability of hazardous waste hy bacterial soil populations. The potential for manufac-

tured gas plant (MGP) soil bacterial populations to degrade naphthalene was

evaluated by the detection of the naphthalene genotype in direct DNA extracts of thesoils and colony hybridization of cultured bacteria. The activity of the naphthalene-

degrading populations was evaluated by 14C02 production from 14C-naphthalene.Direct messenger RNA (mRNA) extraction from MGP soil was evaluated as aninstantaneous measure of catabolic activity in MGP soil. The availability of naphtha-lene within the contaminated soils for bacterial degradation was assessed by mea-

suring the bioluminescent response from a lux-naphthalene catabolic reporterplasmid, pUTK21.

51

DNA extracts of 5 MGP and 1 creosote-contaminated soil hybridized with a nahA

gene probe indicating that the naphthalene genotype was present. 14 C_

Naphthalene mineralization was observed with 14CO 2 rate constants ranging from

0.20 and 2.49h -1. Phenanthrene, anthracene, and benz(a)pyrene were mineralized

also by some of the soils. NAH7-related messenger RNA transcripts were detectable

in the one MGP soil and one creosote-contaminated soil examined. Messenger RNA

analysis of the remaining soils is in progress. Naphthalene bioavailability within the

soil was indicated by a bioluminescent response from the lux-naphthalene construct.

Nucleic acid extractions and hybridizations with gene probes allows a more rapid,

more specific means than traditional kinetic and microbiological techniques of

assessing the presence and activity of the PAH-degrading bacterial population in

PAH contaminated soils.

A Rapid Method for Direct Extraction of DNA from Contaminated Soil and Sedi-

ments Yu-Li Tsai, Ph.D. and Betty H. Olson, Ph.D., Program in Social Ecology, The

University of California

The combined techniques of DNA extraction from polluted environments and

gene probe technology have become a useful tool to monitor particular organisms

contributing to the bioremediation process. A rapid direct extraction of DNA from

contaminated soil and sediments has been developed to overcome the disadvan-

tages of previously published DNA extraction methods. The indigenous organisms

contained in soil/sediment samples were lysed by lysozyme and 3 cycles of

freeze-thawing in the presence of sodium dodecyl sulfate. The lysates were

extracted with phenol-chloroform and followed by isopropanol precipitation to obtain

crude total DNA. In addition to a high recovery rate (>90%), the yields of DNA were

high (38 ug and 12 ug/g net wt. from sediments and soil, respectively). This method

caused minimal shearing of the DNA. The crude DNA could be further purified by an

Elutip - d column if necessary. This method has an additional advantage that only 1

g of sample is required, allowing the analysis of limited sample sizes and the pro-

cessing of many samples in a relatively short period of time.

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On-line, Rea;-time Biosensors for Bioprocesses in Microbial Consortia at the

Institute for Applied Microbiology D.C. White, M.D. Ph.D., D.E. Nivens, M.J.Franklin, N.J.E. Dowling, Ph.D., T.J. Phelps, Ph.D., D.E. Hedrick, D.B. Ringelberg,

Institute for Applied Microbiology, The University of Tennessee, Knoxville

Biofilms formed of consortia of microbes of different physiological types

on surfaces are important in many processes of industrial import. IAM has

developed on-line, non-destructive biomonitoring technologies and high reso-

lution semi-continuous destructive monitoring techniques to follow adhesion,

biofilm formation, community succession, nutritional status and metabolic

activities. Primary work has been directed towards control of heat transfer

resistance and the induction of microbially influenced corrosion (MIC) which is

being increasingly recognized as an extremely important economic and safety

problem for industrial water systems. The development of sufficiently rugged

and accurate monitoring devices by which biofilm formation and activity of

microbial biofilms can be monitored nondestructive'%, directly in water

systems is the goal of this research. This on-line systems would allow the

effective utilization of minimal levels of biocides and inhibitors as well as permitin situ testing of materials for MIC resistance. Several non-destructive technol-

ogies such as the quartz crystal microbalance (QCM), the attenuated total

reflectance-Fourier transforming infrared spectrometer (ATR-FT/IR), and a

genetically engineered bacterium containing the lux gene cassette (developed

in collaboration with G.S. Sayler, of the UTK Center for Environmental Biotech-

nology) in which its bioluminescence can be used to define its presence on

coupons are on-line devices which accurately measure biofilm formation.

These may be correlated to the open circuit potential (OCP), which under

specific conditions correlates with the formation of microbial biofilms and is a

sufficiently rugged electrode for in situ use. Corrosion activity can be esti-

mated by electrochemical impedance spectroscopy (EIS) which is non-

destructive, correlates to microbial biofilm activity, is an accurate monitor of

corrosion, can indicate localized (pitting) corrosion, and is also sufficiently

53

rugged for in situ monitoring. Efforts are presently underway to apply these

technologies to organic waste bioremediation and heavy metal immobilization

by microbial consortia.

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5 FINDINGS

An overall summary of the meeting was stated in the Preface of the published pro-

ceedings of the symposium. 5

The use of biotechnical processes in control of environmental pollution and inhazardous waste treatment is viewed as an advantageous alternative or adduct tophysical chemical treatment technologies. Yet, the development and implementa-tion of both conventional and advanced biotechnologies in efficacious field appli-cations suffer from numerous technical, regulatory and societal uncertainties.

With the application of modern molecular biology and genetic engineering,there is clear potential for biotechnical developments that will lead to break-throughs in controlled and optimized hazardous waste treatment for in situ andunit process use. There is, however, great concern that the fundamental researchbase may not be able to sustain continued technology development.

Some of these issues have been discussed in a fragmented fashion within theresearch and development community. A basic research agenda has been estab-lished to promote a sustainable cross-disciplinary technology base. This agendaincludes developing new and improved strains for biodegradation, improvingbioanalytical methods to measure strain and biodegradation performance, andproviding an integrated environmental and reactor systems analysis approach forprocess control and optimization.

There remains an identified need to promote cross-disciplinary communicationof technology development and application, and to identify choke points thatimpinge on the effective commercial application of the technology. For thesereasons, industrial, federal, and academic partners joined together to sponsor thiscurrent dialogue on moving modern environmental biotechnology from the labora-tory to successful field application. Unlike other efforts to communicate the tech-nology, this symposium was planned to not only identify current practices andstate-of-the-art, but also to identify perceptional and regulatory issues that affectcredible applications and evaluation of the technology. In this regard, we mustacknowledge the concerned foresight of the sponsors of the symposium, Interna-tional Technology Corporation; the American Cyanamid Company; the U.S. AirForce Office of Scientific Research; the University of Tennessee; Waste

5 Sayler, G.S., R.D. Fox, and J. W. Blackburn, Eds., 1991, Environmental Biotechnologyfor Waste Treatment, Plenum Press, New York.

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Management Research and Education Institute; support from the Oak RidgeWaste Management Association; the planning and steering committee and thesymposium participants.

A goal of the symposium was to communicate a broad view of environmentalbiotechnology ranging from conventional practices in biological waste treatment togenetic engineering perspectives in in situ treatment technology. From the begin-ning it was acknowledged that the biology was intimately linked to the environ-mental application and the engineering design in implementing the technology.This major scale-up consideration is the critical technical hurdle in moving thetechnology from the lab to practical field use. In this scale-up, there are major lim-itations in monitoring and controlling biotechnical processes, and these limitationsfurther confound societal and regulatory perception of the credibility of thetechnology.

The outcome of this symposium contributes to identifying applications of funda-mental research in emerging technology and to defining industrial research needs.It is also anticipated that strategies will be forthcoming to overcome concerns ofthe safety and efficacy of the technology. There appear to be numerous opportu-,iiies for environmental biotechnology to contribute to integrated waste manage-ment, but care must be taken to demonstrate reliable technology in order tocapitalize on these opportunities.

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